Could cannabinoids provide a new hope for ovarian cancer patients?

Abstract It is known that gynecological cancers remain a worldwide problem and as shown by the statistics, there is a need for new gynecological cancer treatments. Cannabinoids, the pharmacologically active compounds of the Cannabis sativa plant, have been used for many centuries by individuals as a symptomatic treatment to alleviate pain, nausea, vomiting, and to help stimulate appetite. Research has revealed that cannabinoids also exert anti‐cancer activity such as anti‐proliferative and pro‐apoptotic effects through a variety of mechanisms. There is significant value in the development of these compounds as anti‐cancer therapies in clinical practice as they do not produce the typical toxic side effects that exist with conventional therapies and recent clinical trials have shown their great tolerability by patients at high doses. Cannabinoids can induce psychoactive effects that could limit their progression. Therefore, non‐psychoactive cannabinoids are attracting pharmacological interest due to their inability to produce psychological effects. Recent studies have focussed on non‐psychoactive cannabinoids in ovarian cancer and have revealed promising pre‐clinical results that indicate that these compounds may have potential benefits in the treatment of these cancers. However, there are still unanswered questions and research gaps that need to be addressed. This review summarizes the current understanding of this topic and identifies the current gaps in knowledge that provide a useful direction for future work.


| INTRODUC TI ON
Gynecological cancers are cancers of the female reproductive system and consist of cancers of the cervix, ovaries, vagina, vulva, and uterus. 1 Among gynecological cancers, ovarian cancer has the highest morbidity and mortality rates. 2 Globally, the number of patients diagnosed in 2018 with gynecological cancers was >295 000 for ovarian. In 2018, approximately 185 000 women died from ovarian cancer. 3 It is clear that there are unfavorable outcomes and that today, there are still unmet clinical needs.
Despite improvements in treatment strategies (Table 1), tumor recurrence, drug resistance, and toxic effects remain a major challenge, indicating the need for a new approach to the treatment of ovarian cancer. Recently, cannabinoids have gained attention as potential anti-cancer therapies due to their ability to modulate the intracellular signaling pathways involved in cancer progression. 4 However, the presence of psychoactive effects of cannabinoids could limit their progress in this field, therefore recent studies have highlighted the value of non-psychoactive cannabinoids such as cannabidiol (CBD). 5 CBD has been shown to demonstrate a broad array of anti-carcinogenic properties, such as anti-proliferative action toward breast cancer cells, 6 anti-invasive and anti-metastatic effects on lung cancer cells 7 as well as induction of apoptosis. 8 In this review, the current knowledge on ovarian cancer treatment and the rationale for new therapies are discussed. In addition, this review provides a summary of the endocannabinoid system, the action of cannabinoids in cancer settings, and their anti-tumor properties, as well as the effects of non-psychoactive cannabinoids in ovarian cancer. Finally, recommendations for future work will be included in relation to their potential as a novel gynecological cancer treatment.

| CURRENT CLINI C AL S TATUS OF OVARIAN C AN CER AND R ATI ONALE FOR NOVEL THER APIE S
The current standard of care for patients with ovarian cancer is a combination of optimal cytoreductive surgery and platinum-based chemotherapy. 10 Ovarian cancer usually metastasizes within the peritoneal cavity within the abdomen first and in these cases, surgical debulking is used to inform staging and adjuvant therapy.
Key advances in radical surgery and chemotherapy strategies have led to improved, yet modest, clinical outcomes. Despite this, there remains a significant risk of resistance to drug therapy and tumor recurrence. Due to the site of presentation and the shortage of promising screening tools for early-stage detection, ovarian cancer often presents at a late stage, resulting in a poor 5-year survival rate for these patients even with optimal care. 10 Patients with stage III or IV disease have a 70%-75% chance of recurrence within 2 years of diagnosis. Treatment options are less effective at each recurrence, highlighting the need for novel maintenance therapies (Table 1). 10 Further treatments after relapse are more intense, resulting in increased drug toxicity, drug resistance, and financial burden to patients with poor quality of life. 11 Thus, there is a clear need for new and improved therapies to address the issues that remain with current conventional treatments.

| THE HIS TORY OF C ANNAB INOIDS
The first discovered and most important source of cannabinoids was the Cannabis sativa plant, which has been used as a herbal remedy for centuries. The earliest archeological evidence of the use of medicinal cannabis use was in ancient China where it was recommended for rheumatic pain, constipation, disorders of the female reproductive tract, and malaria, among other conditions. 12 In Western medicine, the use of cannabis was introduced by William B. O'Shaughnessy (an Irish physician) and Jacques-Joseph Moreau (a French psychiatrist) in the mid-19th century. They described positive effects of cannabis preparations on pain, vomiting, convulsions, rheumatism, tetanus, and mental ability. From 1851, cannabis was recognized as a medicine in the United States (US) Pharmacopeia, in the form of tinctures, extracts, and resins. However, at the beginning of the 20th century, cannabis use decreased due to its increased use as a recreational drug, abuse potential, variability in the quality of herbal material, unidentified active compounds, and alternative medications with known efficacy being introduced to treat the same symptoms. 13,14 In 1941, as the result of many legal restrictions, cannabis was considered to be in the same group as other illicit drugs and was removed from the American Pharmacopeia. 14  Endocannabinoids are produced in our body and consist of lipid molecules containing long-chain polyunsaturated fatty acids, amides, esters, and ethers that bind to cannabinoid receptors. 24 Endocannabinoids act mainly as neuromodulators that affect the release of various neurotransmitters in the peripheral and neural tissues. 25 They also play an important role in inflammation, insulin sensitivity, and fat and energy metabolism. 26 Two of the bestcharacterized endocannabinoids are N-arachidonoylethanolamine (AEA-anandamide) and 2-arachidonoylglycerol (2-AG), which are derived from arachidonic acid and affect our mood, appetite, pain sensation, inflammatory response, and memory. 27,28 Synthetic cannabinoids such as the CB 2 synthetic agonist, JWH-133, and the CB 1 and CB 2 agonist, WIN-55 have been widely used as a pharmacological agent, both in vitro and in vivo to obtain a more detailed understanding of cannabinoid action in order to evaluate their potential clinical use. 29,30

| THE ENDOC ANNAB INOID SYS TEM (EC S)
Historically, the two main cannabinoid receptors: cannabinoid receptor 1 (CB 1 ) and cannabinoid receptor 2 (CB 2 ), the main endocannabinoids: AEA-anandamide and 2-AG and the enzymes that produce and degrade these endocannabinoids have been known as ECS. 31 In recent years, further components have widened this origi-

| C ANNAB INOID RECEP TOR S
Insights into the mechanism of action of phytocannabinoids led to the identification of two G protein-coupled receptors, CB 1 and CB 2. 36,37 CB 1 is mainly expressed in the human CNS and is the main receptor responsible for the psychotropic effects of THC. 38,39 Although to a lesser extent, CB 1 receptors are also expressed at peripheral tissue sites where they aid in the regulation of local tissue functions. 39 CB 1 expression has been reported in adipose tissue, skeletal muscle, bone, skin, eye, reproductive system, and several types of cancer cells. 40 A principal role for CB 1 receptors is to inhibit neurotransmitter release. Strongly associated with GABAergic (inhibitory) and glutamergic (excitatory) cells, activation of CB 1 receptors inhibits the release of GABA and glutamate, respectively. 41 This decrease in excitability and neurotransmitter release may underlie some of the psychoactive and anti-convulsant action of cannabinoids.
In contrast, CB 2 receptors are predominantly expressed in peripheral tissues, such as the immune system, where they modulate immunological function, cell migration, and cytokine release. 39,42 However, CB 2 receptor expression has also been detected in the brain, however to a much lower extent compared to the immune system or the level of CB 1 expression. 39  Therefore, it is difficult to determine whether GPR18 is a cannabinoid receptor.

| C ANNAB INOIDS AND THEIR ANTI -TUMOR EFFEC TS
Cannabinoids have an established role in exerting palliative effects in cancer patients and have been used to alleviate nausea, vomiting, pain and to help stimulate appetite. 5 protects against colon tumourigenesis. The study showed that CBG stimulated ROS production, promoted apoptosis, and reduced cell growth in colorectal cancer cells. 77 Table 2 provides a summary of the anti-tumor effects of cannabinoids in different cancers.
The accumulated data show that non-psychoactive cannabinoids such as CBD and CBG have illustrated a range of anti-cancer effects in a multitude of different cancer cell lines. Therefore, CBD and CBG are attracting pharmacological interest due to their nonpsychotropic nature, ability to inhibit cancer cell proliferation, and induction of apoptosis.

| THE ROLE OF C ANNAB INOIDS IN MED IATI ON OF AP OP TOS IS
Apoptosis is a form of programmed cell death that is essential for the development and survival of organisms. 78,79 Defects in the regulation of apoptotic cell death contribute to many diseases, including disorders in which cell accumulation occurs, such as cancer. The molecular machinery responsible for apoptosis has been revealed, uncovering a family of proteases, the caspases, which are accountable for the morphological and biochemical changes that characterize apoptosis. 80   Therefore, it can be concluded that inhibiting the pro-angiogenic Lα-lysophosphatidylinositol/GPR55 pathway may be a promising target against angiogenesis in ovarian cancer. 93 The expression of different components of the ECS is not uniform across all cancers; therefore, pharmacological manipulations of the ECS in further studies allow investigation into the link between the ECS and cancer progression. 89,94 Events in the ovarian cycle are controlled by hormones secreted by the hypothalamus, the anterior pituitary, and the ovaries, collectively known as the hypothalamic-pituitary-gonadal axis. 95 The This would ultimately enable us to establish the therapeutic potential of targeting the estrogen and ECS interaction as an approach for the treatment of gynecological cancers.

| C ANNAB INOIDS IMPLIC ATI ON IN CELL DE ATH THROUG H AP OP TOS IS , CELL C YCLE ARRE S T, AND AUTOPHAGY IN OVARIAN C AN CER
Research has highlighted key proteins and pathways involved in apoptosis that have been altered to evade cell death in ovarian cancer. Through comparing caspase-3 and caspase-8 levels in normal ovary, benign mass, and ovarian cancer, in vitro studies showed that caspase-3 and caspase-8 levels were lower in the benign mass and malignant group compared to the normal ovary group. 107 Similarly, another study also showed that ovarian cancer tumors had low levels of caspase-8 and were associated with shorter overall survival compared to tumors from patients that had high levels of caspase-8. 108 Therefore, since ovarian tumors have been shown to exhibit lower levels of these caspases, activating this pathway and thus increas-  115 In ovarian cancer cells, enforcing cyclin E1 expression stimulates cell proliferation, 116 and over-expression of cyclin E1 has been linked to the development of chemo-resistance in ovarian cancer. 117,118 It has been shown that ovarian tumors with elevated cyclin E1 levels often exhibit higher CDK2 expression. 119,120 Studies show that the abundance of cyclin E1 correlates with tumor progression in patients with ovarian cancer. [121][122][123][124] Mutations in the tumor suppressor gene, p53, have been detected in all histological types of epithelial ovarian cancer, and serous carcinomas, have been detected at higher frequencies. Alterations in the p53 network represent up to 96% of patients with high-grade serous ovarian carcinoma. 125 The loss of p53 function is another exploited mechanism that ovarian cancer cells deploy to inhibit cell cycle arrest and apoptosis. Dysregulation of the cell cycle signaling pathway CDK4/6cyclin D/p16-Rb is one of the most common abnormalities in human cancer. 126 Studies have identified that in ovarian cancer, p16 expression is most commonly altered due to promoter methylation. [127][128][129] Overexpression of cyclin D1 has been described in ovarian cancer tumors and has been associated with a more aggressive tumor phenotype and poor prognosis. 130 Mutations of the Rb gene have been reported in ovarian cancers. 131,132 Together, these altered proteins and signaling pathways involved in the cell cycle regulation contribute to uncontrolled cell proliferation in ovarian cancer by avoiding cell cycle arrest.
Autophagy, from the Greek, meaning self-eating refers to a cellular process committed to the lysosomal degradation of selfconstituents. 133 Dysregulation of autophagy plays a significant role in the pathogenesis and resistance to radiotherapy and chemotherapy in ovarian cancer. A plethora of signaling pathways and proteins whose expression has been found altered in ovarian cancers have an impact on autophagy. 134 For example, alterations in LC3, a component of the autophagy machinery. Compared to benign tissues and borderline ovarian tumors, highly malignant ovarian cancer cells were shown to express very low levels of LC3. 135 Another example is an alteration in the signaling pathway, PI3K-AKT-mammalian target of rapamycin (mTOR). In ovarian cancer patients, a hyperactive state of mTOR has been associated with a poor prognosis. 136 There is also sustained up-regulation of the PI3K-AKT-mTOR pathway in ovarian cancers that results in increased suppression of the autophagic process. 137,138 Moreover, deletion of the gene that encodes Beclin-1, another component of the autophagy machinery, has been identified in ovarian cancers. 139 The expression of Beclin-1 has been shown to be dramatically higher in benign and borderline ovarian tumors than those in malignant ovarian cancers. 135  the ability to evade cell death in ovarian cancer ( Figure 1).
F I G U R E 1 A summary of the altered signaling pathways and proteins leading to evasion of cell death via apoptosis, cell cycle arrest, and autophagy in ovarian cancer that are affected by cannabinoids.

| CONCLUSION AND FUTURE DIRECTION
The

AUTH O R CO NTR I B UTI O N S
Both authors contributed to all aspects of writing.

ACK N O WLE D G E M ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Not applicable.

E TH I C S S TATEM ENT
Not applicable.